CD147‐K148me2‐Driven Tumor Cell‐Macrophage Crosstalk Provokes NSCLC Immunosuppression via the CCL5/CCR5 Axis

Abstract Immunosuppression is a major hallmark of tumor progression in non‐small cell lung cancer (NSCLC). Cluster of differentiation 147 (CD147), an important pro‐tumorigenic factor, is closely linked to NSCLC immunosuppression. However, the role of CD147 di‐methylation in the immunosuppressive tumor microenvironment (TME) remains unclear. Here, di‐methylation of CD147 at Lys148 (CD147‐K148me2) is identified as a common post‐translational modification (PTM) in NSCLC that is significantly associated with unsatisfying survival outcomes among NSCLC sufferers, especially those in the advanced stages of the disease. The methyltransferase NSD2 catalyzes CD147 to generate CD147‐K148me2. Further analysis demonstrates that CD147‐K148me2 reestablishes the immunosuppressive TME and promotes NSCLC progression. Mechanistically, this modification promotes the interaction between cyclophilin A (CyPA) and CD147, and in turn, increases CCL5 gene transcription by activating p38‐ZBTB32 signaling, leading to increased NSCLC cell‐derived CCL5 secretion. Subsequently, CD147‐K148me2‐mediated CCL5 upregulation facilitates M2‐like tumor‐associated macrophage (TAM) infiltration in NSCLC tissues via CCL5/CCR5 axis‐dependent intercellular crosstalk between tumor cells and macrophages, which is inhibited by blocking CD147‐K148me2 with the targeted antibody 12C8. Overall, this study reveals the role of CD147‐K148me2‐driven intercellular crosstalk in the development of NSCLC immunosuppression, and provides a potential interventional strategy for PTM‐targeted NSCLC therapy.


Introduction
The tumor microenvironment (TME) is a highly intricate environment composed of inflammatory cells, fibroblasts, and the extracellular matrix and is closely linked to complex biological behaviors in tumors. [1][8] Unlike M1 phenotype-mediated anti-tumor immunity which involves antigen presentation and pro-inflammatory cytokine secretion, M2-TAMs are characterized by high expression of inhibitory molecules, such as PD-L1 and Tim3, which inhibit the activities of cytotoxic T cells and natural killer cells that can eradicate tumors and remodel the immunosuppressive TME during tumor evolution, leading to malignant behaviors. [6,9]Although many studies have investigated the carcinogenic effects of M2-TAMs in multiple cancers, the detailed mechanisms of immunosuppressive TME reconstruction induced by interactions between tumor cells and M2-TAMs have not yet been fully elucidated.
As a common post-translational modification (PTM), protein methylation, including histone and non-histone methylation, has a critical effect on tumor progression by regulating protein-protein interactions, protein-DNA interactions, protein subcellular localization, and protein stability [10] and is closely associated with M2-TAM-mediated immunosuppressive TME remodeling. [11,12]For histones, H3K4 tri-methylation increases tumor cell-derived CCL2 secretion by activating CCL2 transcription, which promotes M2-TAM recruitment and activation, leading to lymphatic metastasis in bladder cancer. [13]G9A-mediated H3K9 di-methylation inhibits Fbxw7 transcription in glioma stem cells and subsequently promotes immune suppression by increasing the infiltration of M2-TAMs in the TME. [14]In addition, non-histone methylation is involved in M2-TAM-mediated immunosuppression.For example, MCT1 tri-methylation mediated by the methyltransferase SETDB1 sustains MCT1 stabilization and in turn promotes tumor glycolysis and M2-like polarization of TAMs in colorectal cancer, which contributes to tumor immunosuppression. [15]These studies indicate that abnormal methylation exacerbates the immunosuppressive TME by regulating tumor cell-macrophage intercellular crosstalk, suggesting that targeting methylation or methyltransferases in the TME may be a strategy for tumor treatment.
[18][19] As the role of CD147 in tumor progression has been further elucidated, the PTMs of CD147 have been extensively investigated owing to their diversity and heterogeneity.Among them, di-methylation is a relatively common PTM of CD147 and is found at multiple lysine residues.The di-methylation of CD147-K234 catalyzed by the methyltransferase KMT5A is highly expressed in non-small cell lung cancer (NSCLC), which drives tumor glycolysis and lactate export by enhancing the interaction between CD147 and MCT4, and promotes tumor progression. [20]However, compared to that in para-carcinoma tissues, SETDB1-mediated di-methylation of CD147-K71 is substantially decreased in NSCLC, increasing tumor cell sensitivity to chemotherapy-induced apoptosis. [21]These studies show that different di-methylations of CD147 have distinct biological functions in mediating tumor progression.However, the precise mechanisms by which CD147 di-methylation drives M2-TAM-mediated tumor immunosuppression are still unclear.
In our study, we discovered that methyltransferase NSD2mediated CD147 di-methylation at Lys148 (CD147-K148me2) was significantly greater in NSCLC tissues than in paracarcinoma tissues.This targeted modification increased NSCLC cell-derived CCL5 secretion via CyPA-CD147-p38-ZBTB32 signaling, which promoted NSCLC immunosuppression via CCL5/CCR5 axis-dependent tumor cell-macrophage crosstalk.Our study demonstrates the positive effect of CD147 di-methylation on the formation of an immunosuppressive TME and provides a promising interventional strategy for NSCLC therapeutic regimens by targeting PTMs.

CD147 Di-Methylation at Lys148 Is Increased in NSCLC Patients
CD147 is a transmembrane protein that plays a crucial role in tumor progression.The biological function of CD147 mainly depends on its extracellular domain (ECD).Interactions between ligands or extracellular signals and CD147-ECD initiate a series of intracellular biological chemical reactions and subsequently trigger signal transduction.A previous study identified a panel of lysine residues of CD147-ECD modified by dimethylation in NSCLC samples. [20]To screen for differential dimethylation of CD147-ECD between NSCLC and para-carcinoma tissues, we collected 20 pairs of NSCLC and para-carcinoma tissues and measured the abundance of CD147 di-methylation at the residues of the CD147-ECD using liquid chromatographytandem mass spectrometry (LC-MS/MS).The di-methylation of CD147 at Lys75, 111, 127, and 148 was significantly greater in the NSCLC tissues than in the para-carcinoma tissues, while the di-methylation of CD147 at Lys71 was lower in the NSCLC tissues than in the para-carcinoma tissues (Figure S1A, Supporting Information).The ratio of the modified peptide-spectrum matches (PSMs) to unmodified PSMs for the peptide K148 was the highest among all the di-methylated lysine residues (Figure S1B, Supporting Information).Hence, we chose CD147-K148me2 for further investigation (Figure S1C,D, Supporting Information).
To investigate the expression of CD147-K148me2 in NSCLC samples, we developed a monoclonal antibody (12C8) specifically targeting CD147-K148me2 by immunizing mice with the CD147-K148me2 peptide (Figure S2A, Supporting Information).The subtype of the 12C8 antibody was IgG1 (Figure S2B, Supporting Information).Dot blot and enzyme-linked immunosorbent assays (ELISAs) revealed the strong specificity of 12C8 against CD147-K148me2; this antibody exclusively binds to the peptide CD147-K148me2 without cross-reactivity with the peptide CD147-K148 or peptides containing other di-methylated lysine residues of CD147 (Figure S2C-E, Supporting Information).Then, 12C8 was used to determine the CD147-K148me2 levels in NSCLC and para-carcinoma tissues by immunohistochemical (IHC) staining, and CD147 itself was also assessed as a reference.According to the expression levels of CD147-K148me2 and CD147, the NSCLC tissues were divided into four groups, namely, negative (-), weak (+), moderate (++), strong (+++).The percentages of CD147-K148me2-positive tissues were 65% in lung adenocarcinoma (LUAD) and 71% in lung squamous cell carcinoma (LUSC); these values were slightly lower than those of CD147, and both molecules were barely detectable in paracarcinoma tissues (Figure 1A-C; Figure S3A-C, Supporting Information).Then, negative (-) and weak (+) samples were classified as the low group, while moderate (++) and strong (+++) samples were classified as the high group.Clinical analysis revealed that the expression of CD147-K148me2 was closely linked to gender, tumor size, and pathological staging in LUAD patients (Table S1, Supporting Information), while no significant correlation was found in LUSC patients (Table S2, Supporting Information).Moreover, the expression of CD147 was associated with tumor size and N staging in LUSC patients, while no correlation was found in LUAD patients (Tables S3,4, Supporting Information).Kaplan-Meier analysis revealed that high expression of both CD147-K148me2 and CD147 was significantly positively associated with poor overall survival in NSCLC patients (Figure 1D-F; Figure S3D-F, Supporting Information).For patients with NSCLC in the earlier stages (AJCC stages I-II), a high CD147 level was associated with unsatisfactory clinical outcomes, whereas a high CD147-K148me2 level worked for NSCLC patients in the advanced stages (AJCC stages III-IV) (Figure 1G,H; Figure S3G,H, Supporting Information).These findings reveal that CD147-K148me2 is highly expressed in NSCLC and has prognostic value for NSCLC patients, especially those in the advanced stages of the disease.

The Methyltransferase NSD2 Acts on Lys148 of CD147 to Generate CD147-K148me2
Given the importance of CD147-K148me2 in NSCLC progression, we investigated the specific methyltransferase for CD147-K148 to further elucidate this modification process.siR-NAs targeting the corresponding methyltransferases (SETD1A, SETDB1, SETD6, SETD7, G9A, EZH2, SUV39H2, NSD1, and NSD2) were synthesized and transfected into H460 cells.Then, the expression of CD147 and CD147-K148me2 was determined by western blot.Interestingly, the transfection of siRNAs did not affect CD147 expression but specifically reduced the level of CD147-K148me2 in the NSD2 knockdown groups (Figure 2A; Figure S4A,B, Supporting Information).A similar result was also obtained in A549 cells (Figure 2A), suggesting that NSD2 is a potential candidate methyltransferase for CD147-K148.The docking model showed that the di-methylation site Lys148 of CD147 was positioned toward the catalytic pocket of the methyltransferase NSD2, where the methyl donor SAM was located (Figure 2B).The distance between the side chain of Lys148 and SAM was 11.9 Å, indicating close proximity for the methylation reaction.The free energy of binding (ΔG) of the complex was calculated to be −8 kcal mol −1 by PRODIGY, suggesting a stable and favorable interaction.These results suggest a possible catalytic mechanism for the di-methylation of CD147 by NSD2.Moreover, co-localization of CD147 and NSD2 was observed in NSCLC tissues (Figure 2C), and a co-immunoprecipitation (co-IP) assay demonstrated an interaction between CD147 and NSD2 in A549 and H460 cells (Figure 2D).Then, stable CD147 knockdown cell lines (A549/H460-shCD147) were generated by lentiviral infection (Figure S5A,B, Supporting Information), and lentiviruses containing wild-type CD147 (CD147-WT) and mutant CD147 (CD147-K148R) were transfected into A549/H460-shCD147 cells to generate CD147-reexpressing cell lines (A549/H460-rWT and rK148R, respectively) (Figure S5C,D, Supporting Information).Subsequently, the CD147-NSD2 interaction was investigated in the A549/H460-rWT and rK148R cell lines.Compared to that in the rWT cells, a weakened interaction between CD147 and NSD2 was found in the rK148R cells (Figure 2E).NSD2 overexpression increased the CD147-K148me2 levels in A549 and H460 cells (Figure 2F).NSD2 was also introduced to the rWT and rK148R cells, and the results demonstrated that NSD2 increased CD147-K148me2 levels in the rWT cells, while no change was observed in the rK148R cells (Figure 2G).In vitro methyltransferase assays revealed that the peptide K148 and His-CD147-WT protein were methylated by NSD2 in methyltransferase buffer, while this modification was not found in the His-CD147-K148R group (Figure 2H,I).These findings indicate that the methyltransferase NSD2 catalyzes the Lys148 residue of CD147 to generate CD147-K148me2.

CD147-K148me2 Promotes CCL5 Secretion and M2-Like TAM Migration in NSCLC
To investigate the role of CD147-K148me2 in NSCLC progression, we performed proliferation and migration assays, and the results revealed no obvious differences between the rWT and rK148R groups (Figure S6A-D, Supporting Information).Then, we conducted RNA-seq analysis using CD147-WT (WT)-and CD147-K148R (Mut)-overexpressing A549 cells, with A549 cells transfected with a mock plasmid (NC) serving as the control (Figure 3A).Genes with a fold change >1.5 and P<0.05 were defined as differentially expressed genes (DEGs).Among the DEGs, CCL5 was the most significant DEG between the WT and Mut groups and was also among the top 10 DEGs between the NC and WT groups (Figure 3B).These results indicated that CD147-K148me2 promoted CCL5 secretion in NSCLC cells.Western blot analysis revealed a decreasing trend in CD147-K148me2 and CCL5 expression in the rK148R group compared with the rWT group (Figure 3C), and this result was also verified in A549 and H460 cells by RT-PCR (Figure 3D).Since CCL5 is a secreted protein, the content of CCL5 in the culture medium was determined by ELISAs, and similar results were obtained (Figure 3E).Taken together, these findings indicate that this modification promotes CCL5 generation in NSCLC cells.
Subsequently, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were performed using the abovementioned RNA-seq data.The top 20 GO terms of DEGs in the WT versus Mut and NC versus WT groups included "cell chemotaxis", "macrophage chemotaxis", "immune response", "response to cytokine", and "cytokine-mediated signaling pathway" (Figure S7A,B, Supporting Information).GSEA also identified "chemokine activity", "CCR chemokine receptor binding", "monocyte chemotaxis", and "response to chemokine" (Figure S7C,D, Supporting Information).Similar results were obtained via KEGG analysis (Figure S8A-D, Supporting Information).These results indicate that CD147-K148me2 is closely related to immune cell chemotaxis and the immune response.
CCL5 is an important chemotactic cytokine that promotes immune cell migration via the CCL5/CCR5 axis. [22]Then, the NSCLC single-cell RNA sequencing data were analyzed, and CCR5 was found to be present in monocytes and macrophages (Figure S9A,B, Supporting Information).Data from the TISIDB database showed a significant correlation between CCL5 expression and macrophage infiltration in LUAD and LUSC (Figure S9C, Supporting Information).Previous studies showed that the CCL5/CCR5 axis contributed to tumor progression by promoting The interaction between CD147 and NSD2 was confirmed in A549 and H460 cells by co-IP assays, and IgG was used as a negative control.E) The interaction between CD147 and NSD2 was analyzed in the rWT and rK148R cells (A549 and H460) by co-IP assays, and IgG was used as a negative control.F) The expression of CD147, NSD2, and CD147-K148me2 was determined in the NSD2-overexpressing cells (A549 and H460).G) The expression of CD147, NSD2, and CD147-K148me2 was determined in the NSD2-overexpressing rWT and rK148R cells (A549 and H460).H) An in vitro methyltransferase assay was performed with the K148 peptide and recombinant NSD2 protein co-incubated in methyltransferase buffer containing the methyl donor SAM.The di-methylation of the K148 peptide was detected by an anti-CD147-K148me2 antibody (12C8).I) An in vitro methyltransferase assay was performed with recombinant NSD2 protein and His-CD147-WT or His-CD147-K148R co-incubated in methyltransferase buffer containing the methyl donor SAM.The di-methylation of the CD147 protein was detected by an anti-CD147-K148me2 antibody (12C8).
Subsequently, the chemotaxis of M2-TAMs mediated by CD147-K148me2-induced CCL5 secretion was evaluated by a Transwell assay.The THP-1 cells were differentiated into M2-like macrophages with increased expression of CD163 and CD206 (Figure S10A,B, Supporting Information).Chemotaxis assays C) The expression levels of CD147, CD147-K148me2, and CCL5 were determined in the rWT and rK148R cells (A549 and H460) by western blot analyses.D) The mRNA level of CCL5 was determined in the rWT and rK148R cells (A549 and H460) by RT-PCR (*P < 0.05; **P < 0.01; n = 3).E) CCL5 levels in revealed that M2-like macrophages exhibited greater migration in the rWT group than in the rK148R group (Figure 3F,G; Figure S10C,D, Supporting Information).The addition of CCL5 antibody to the culture medium inhibited M2-like macrophage migration in the rWT group, whereas no significant alteration was observed in the rK148R group (Figure 3H,I; Figure S10E,F, Supporting Information).Similarly, CCR5 inhibition in Maraviroc-treated M2-like macrophages yielded comparable results in both the rWT and rK148R groups (Figure 3J,K; Figure S10G,H, Supporting Information).These findings indicate that CD147-K148me2 facilitates CCL5 secretion and increases M2-like TAM migration via CCL5/CCR5 axis-mediated cellular crosstalk, suggesting that CD147-K148me2 contributes to the immunosuppressive TME in NSCLC.

CD147-K148me2 Facilitates CCL5 Secretion in NSCLC Cells by Activating CyPA-CD147-p38 Signaling
The above findings revealed a positive correlation between CD147-K148me2 levels and CCL5 secretion in NSCLC cells.However, the underlying mechanism responsible for the CD147-K148me2-mediated increase in CCL5 was still unknown.Previous studies have reported that CD147, an important inflammation-related molecule, facilitates the secretion of cytokines and chemokines via CyPA/CD147-activated intracellular signal delivery, including ERK1/2, p38, and JNK mitogenactivated protein kinase (MAPK) signaling pathway. [24,25]Thus, we propose that CD147-K148me2 promotes CCL5 secretion via CyPA/CD147-activated MAPK signaling.Accordingly, we investigated the effect of CD147-K148me2 on the interaction between CyPA and CD147.The Lys148 residue is located in the loop region that connects two  strands of CD147, and the B-factor distribution based on the CD147 crystal structure showed that this loop had the highest B-factor value, indicating its high structural flexibility (Figure 4A).To further explore the structural mechanism by which CD147-K148me2 leads to altered binding with CyPA, we constructed structural models of CyPA in complex with Lys148di-methylated and unmethylated CD147 (Figure S11A, Supporting Information) using a strategy based on molecular docking and short-time dynamic optimization.As shown in Figure 4B, the side chain of unmodified CD147-K148 formed a polar interaction with CD147-E155 on the opposite side of the loop, which stabilized the loop region to some extent.This di-methylation caused an increase in the volume of the Lys148 side chain, disrupted the polar interaction with CD147-E155, and thereby increased the overall flexibility of the loop, which facilitated more extensive interactions with CyPA.Further analysis revealed that compared with the unmodified CD147-CyPA complex, the di-methylated CD147-CyPA complex formed more hydrogen bonds at the interface and had a larger interaction area and a lower free en-ergy (Figure 4C; Figure S11B,C, Supporting Information).These findings indicated that this di-methylation increased the binding capacity of CyPA for CD147.Co-IP assays were also conducted to identify the interaction of the two proteins.Compared to that in the rK148R group, the CyPA-CD147 interaction increased in the rWT group (Figure 4D), which verified the structural mechanism by which CD147-K148me2 increased the affinity of CyPA for CD147.
Subsequently, recombinant CyPA protein was added to the culture medium of the rWT and rK148R cells, and the expression level of CCL5 in the rWT group significantly increased under CyPA stimulation, while no change was observed in the rK148R group (Figure 4E).Similar results were also obtained in ELISAs of secreted CCL5 in culture medium (Figure 4F).Moreover, the activation of MAPKs was identified by western blot, and the results showed that CyPA stimulation increased the phosphorylation of p38, while little change in the phosphorylation of ERK1/2 and JNK was found in the rWT group.However, the phosphorylation of ERK1/2, p38, and JNK was not obviously altered in the rK148R group under CyPA stimulation (Figure 4G; Figure S10I, Supporting Information).In A549 and H460 cells, CyPA promoted the phosphorylation of p38, which was inhibited by 12C8 blockade (Figure 4H).Similarly, the CyPA-mediated increase in CCL5 levels was inhibited by a p38 inhibitor (SB203580) (Figure 4I).These findings indicate that CD147-K148me2 promotes the CyPA-CD147 interaction and increases CCL5 secretion by activating p38 signaling.A chemotaxis assay was also conducted in rWT and rK148R cells after CyPA addition, and the results showed that CyPA stimulation increased the migration of M2-like macrophages in the rWT group, while no significant change was found in the rK148R group (Figure 4J-M).Taken together, these findings indicate that CD147-K148me2 facilitates CCL5 secretion in NSCLC cells and increases M2like macrophage migration by activating CyPA-CD147-p38 signaling.
Then, the binding sites of ZBTB32 to the CCL5 gene promoter were analyzed with a relative profile score threshold >90% using JASPAR.The sequence of the CCL5 gene promoter (−387bp-TTTACAGTGT-−378 bp) was considered a candidate binding site.A firefly luciferase reporter vector containing the CCL5 promoter (−1000-−1) was created and transfected into HEK293T cells with pRL-TK with renilla luciferase and the plasmid ZBTB32 or its control.The dual-luciferase reporter assay showed that the CCL5 gene promoter exhibited a greater luciferase signal in the ZBTB32 group than in the control group (Figure 5F,G).To identify the specific binding site of ZBTB32 with the CCL5 gene promoter, we generated truncated sequences of the CCL5 gene promoter, and the results showed that the luciferase signal decreased significantly in the truncated plasmid (−300-−1) group, suggesting that the potential binding site was probably located at positions −400-−300 of the CCL5 gene promoter (Figure 5H).The predicted binding sequence of the CCL5 gene promoter (−387bp-TTTACAGTGT-−378 bp) was deleted and mutated to generate the new plasmids CCL5-Del (−400-−1) and CCL5-Mut (−400-−1), respectively (Figure 5I).Compared to those in the CCL5-WT group, the luciferase signals decreased significantly in both the CCL5-Del and CCL5-Mut groups (Figure 5J), indicating that ZBTB32 interacted with this region.These findings indicate that the −387-−378 region of the CCL5 gene promoter is important for the binding of ZBTB32.
To validate the regulatory effect of CD147-K148me2 on ZBTB32, we examined the expression of ZBTB32 and CCL5 in rWT and rK148R cells with or without CyPA stimulation.The results showed that CyPA supplementation increased the expression of both ZBTB32 and CCL5 in the rWT group, while no obvious change was observed in the rK148R group (Figure 6A).ZBTB32 knockdown in A549 and H460 cells inhibited the ex-pression of CCL5, and this change was not reversed by CyPA supplementation (Figure 6B).In addition, the administration of SB203580 inhibited the CyPA-mediated upregulation of ZBTB32 and CCL5 in A549 and H460 cells (Figure 6C).In the rWT group, SB203580 inhibited ZBTB32 and CCL5 expression by affecting p38 signaling.Supplementation with a p38 activator (U-46619) reversed the expression of ZBTB32 and CCL5 in the rK148R cells (Figure 6D).Immunofluorescence staining showed that ZBTB32 expression in the cell nucleus was greater in the rWT group than in the rK148R group (Figure 6E), and these results were also verified by nuclear and cytoplasmic protein extraction assays (Figure 6F).Meanwhile, CyPA stimulation promoted ZBTB32 expression and facilitated its translocation to the nucleus (Figure 6F).Taken together, these findings indicate that the CyPA-CD147-K148me2 interaction increases CCL5 secretion by activating the p38-ZBTB32 axis in NSCLC cells.

CD147-K148me2 Promotes NSCLC Progression by Inducing M2-Like TAM Infiltration, Which Is Inhibited by an Anti-CD147-K148me2 Antibody
To further investigate the role of CD147-K148me2 in NSCLC progression, we performed in vivo analyses with a cell linederived xenograft (CDX) model.Given the high similarity between human NSD2 and mouse NSD2, lentiviruses encoding human CD147 (WT or K148R) were introduced into mouse LLC cells to generate LLC-WT/K148R cells (Figure 7A).Western blot analysis showed that the levels of CD147-K148me2 and CCL5 were greater in the WT group than in the K148R group (Figure 7B), indicating that mouse NSD2 catalyzes the generation of CD147-K148me2 from human CD147 and that this modification increased the level of CCL5 in LLC cells.Therefore, the LLC-WT/K148R cell lines were suitable for in vivo testing.For the CDX model, the mice were injected subcutaneously with LLC-WT (n = 6) or LLC-K148R (n = 6) cells.At the experimental endpoint, the tumor masses were collected (Figure 7C), and no significant difference in body weight was observed between the WT and K148R groups (Figure 7D).However, the tumor volume in the WT group was greater than that in the K148R group (Figure 7E), and a similar trend was observed for tumor weight in the two groups (Figure 7F).IHC staining revealed a greater percentage of Ki-67+ cells in the WT group than in the K148R group (Figure 7G,H).Multi-color immunofluorescence staining revealed high infiltration of F4/80+CD206+ cells in the WT group, indicating that CD147-K148me2 promoted acid residues involved in hydrogen bond formation are shown as sticks.Hydrogen bonds are represented by yellow dashed lines.D) The interaction between CD147 and CyPA was analyzed in the rWT and rK148R cells (A549 and H460) by co-IP assays, and IgG was used as a negative control.E) CCL5 expression was determined in rWT and rK148R cells (A549 and H460) with or without recombinant CyPA stimulation by western blot.F) CCL5 levels in the culture media of the rWT and rK148R cells (A549 and H460) treated with or without recombinant CyPA were analyzed by ELISAs (***P < 0.001; ns, not significant; n = 4).G) The levels of ERK1/2, p-ERK1/2, p38, p-p38, JNK, and p-JNK were determined in the rWT and rK148R cells (A549 and H460) with or without recombinant CyPA stimulation by western blot.H) The levels of p38 and p-p38 were determined in the A549 and H460 cells with or without recombinant CyPA stimulation or 12C8 blockade, and IgG was used as a negative control.I) CCL5 levels were analyzed in the A549 and H460 cells treated with or without recombinant CyPA or SB203580 by western blot.J,K) A cell chemotaxis assay was conducted by co-incubation of M2-like macrophages and A549-rWT or A549-rK148R cells with or without recombinant CyPA stimulation, scale bar, 200 μm (J), and the quantitative analysis was conducted using three independent experiments (*P < 0.05; ns, not significant; n = 3) (K).L,M) A cell chemotaxis assay was conducted by co-incubation of M2-like macrophages and H460-rWT or H460-rK148R cells with or without recombinant CyPA stimulation, scale bar, 200 μm (L), and the quantitative analysis was conducted using three independent experiments (**P < 0.01; ns, not significant; n = 3) (M).A) The transcription factors associated with CCL5 expression were screened in the WT versus Mut and NC versus WT groups using RNA-seq analysis.Thirteen DEGs were shared between the two groups, and ZBTB32 was the only transcription factor.B) The correlation between ZBTB32 and CCL5 expression was analyzed in the NC and WT groups using RNA-seq data (R = 0.96, P = 0.0019).C) The correlation between ZBTB32 and CCL5 expression was analyzed in the WT and Mut groups using RNA-seq data (R = 0.91, P = 0.012).D) CCL5 expression was determined in the ZBTB32-overexpressing cells (A549 and H460) by western blot.E) CCL5 expression was determined in the ZBTB32 knockdown cells (A549 and H460) by western blot.F) The binding site of ZBTB32 to the CCL5 gene promoter was analyzed with a relative profile score threshold >90% using JASPAR.G) Dual-luciferase reporter assays were performed after co-transfecting of the pGL3-Basic-CCL5 promoter (−1000-−1), pRL-TK, and plasmid ZBTB32 or its control into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; n = 3).H) Dual-luciferase reporter assays were performed after co-transfecting of pGL3-Basic with different truncated sequences of the CCL5 gene promoter, pRL-TK, and the ZBTB32 plasmid into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; ns, not significant; n = 3).I) The predicted binding sequence of the CCL5 gene promoter (−387bp-TTTACAGTGT-−378 bp) was deleted and mutated to generate the new plasmids CCL5-Del (−400-−1) and CCL5-Mut (−400-−1), respectively.J) Dual-luciferase reporter assays were performed after co-transfecting of CCT-WT (CCL5-Del/CCL5-Mut) with pRL-TK and the plasmid ZBTB32 or its control into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; ns, not significant; n = 3).M2-like TAM infiltration (Figure 7I,J).The expression of ZBTB32 and CCL5 was also determined by western blot analysis using tumor masses, and the mutation of di-methylated Lys148 inhibited ZBTB32 and CCL5 expression (Figure 7K,L), which was consistent with the results in A549 and H460 cells.These findings demonstrate that CD147-K148me2 promotes NSCLC progression by increasing M2-like TAM infiltration.
To investigate the efficacy of 12C8 in tumor treatment, we performed an in vivo test by intravenous administration of 12C8 (3 mg kg −1 ) three times, and an equal amount of IgG was used as a control (Figure 8A).Following therapy, the tumor masses were collected (Figure 8B), and the body weights were not significantly different between the IgG and 12C8 groups (Figure 8C).Compared to the control, the administration of 12C8 significantly reduced the volume and weight of the tumor masses (Figure 8D,E).IHC staining demonstrated that the percentage of Ki-67+ cells in the IgG group was greater than that in the 12C8 group (Figure 8F,G), suggesting that 12C8 treatment significantly inhibited tumor growth.Multi-color immunofluorescence staining was performed using slides from both the IgG and 12C8 groups.In contrast to that in the IgG group, the percentage of F4/80+CD206+ cells was lower in the 12C8 group (Figure 8H,I), suggesting that 12C8 treatment significantly suppressed M2like TAM infiltration in tumor tissues.These findings indicate that targeting CD147-K148me2 is a potential strategy for tumor treatment and lays the foundation for PTM-targeted tumor therapy.

Discussion
NSCLC is a common disease that threatens human health.Identifying novel factors associated with NSCLC progression will directly contribute to tumor prognosis and treatment.Advances in proteomics research and LC-MS/MS technology have led to increased focus on PTMs, and these PTMs have gained prominence as potential neoantigens or pathogenic factors that accelerate tumor evolution.][29] In this study, the di-methylation of CD147 at Lys148, which is catalyzed by the methyltransferase NSD2, was identified as a new tumor biomarker for NSCLC.Compared to that in para-carcinoma tissues, CD147-K148me2 was highly expressed in NSCLC tissues.High CD147-K148me2 levels were closely associated with poor overall survival in NSCLC patients, especially in patients with advanced-stage NSCLC, possibly because of the high expression of NSD2 in this stage.Protein methylation plays a crucial role in tumor development and is involved in diverse biological processes, especially regulating protein-protein interactions that contribute to abnormal signal transduction and disease development. [30,31]CyPA and CD147 are regarded as essential ligand and receptor that activate multiple oncogenic signaling pathways, and agents that interfere with the interaction between CD147 and CyPA have the potential to inhibit tumor progression. [25]In our study, the dimethylation of CD147 at Lys148 had a potential influence on protein conformation, which increased the binding capacity of CyPA to CD147, and in turn activated the p38-ZBTB32 signaling, leading to increasing NSCLC cell-derived CCL5 secretion.These findings identify the role of CD147-K148me2 in enhancing CyPA-mediated intracellular signal transduction and illustrate the pathogenesis of the CyPA/CD147 complex in tumor progression from the perspective of PTMs.
It has been reported that the immunosuppressive TME determines the anti-tumor immune response to immunotherapy to some extent, [32] which is usually caused by the intercellular crosstalk between immune cells and immune cells or between immune cells and other cells in the TME.In NSCLC, tumor cells educate macrophages to express MARCO and acquire an immunosuppressive phenotype by secreting IL-37, which diminishes CD8+ T cell activities and killing capacity. [33]Cancerassociated fibroblasts interact with tumor-infiltrating immune cells, reshaping the immunosuppressive TME via the secretion of different chemokines, cytokines, and other effector molecules. [34]n our study, we found that CD147-K148me2-mediated CCL5 increase in NSCLC cells facilitated intercellular crosstalk between tumor cells and M2-TAMs via the CCL5/CCR5 axis, leading to the infiltration of M2-TAMs in NSCLC and tumor progression.Hence, the disruption of abnormal intercellular crosstalk in the TME is beneficial to reverse the immunosuppressive TME and enhance anti-tumor immune response of tumor patients.
[37][38][39] In our study, a specific antibody 12C8 targeting CD147-K148me2 was generated by the immunization with di-methylated peptide.Importantly, the administration of 12C8 inhibited CD147-K148me2-mediated CCL5 increase and the CCL5/CCR5 axis-dependent intercellular crosstalk between tumor cells and macrophages, which decreased the infiltration of M2-TAMs in tumor tissues and showed a notable anti-tumor effect in in vivo tests.These findings demonstrate the contribution of CD147-K148me2 to NSCLC immunosuppression and indicate that this modification is a potential target for NSCLC treatment from the perspective of eradicating tumor immunosuppression, which is expected to increase the effect of cancer immunotherapy for patients with NSCLC.
In summary, our study identifies an important PTM, CD147-K148me2, in NSCLC and reveals the role of CD147-K148me2mediated intercellular crosstalk in NSCLC immunosuppression and progression, suggesting that targeting CD147-K148me2 is a potential interventional strategy for NSCLC therapy.
The mice (C57BL/6J, female, 6-8 weeks old, Beijing HFK Bioscience Co., Ltd.) were injected subcutaneously with LLC-WT (2 × 10 6 cells/mouse, n = 6) or LLC-K148R cells (2 × 10 6 cells/mouse, n = 6).The body weight and tumor volume were measured every two days.At the experimental endpoint, the tumor masses were collected to extract total proteins, which were used to assess the expression of ZBTB32 and CCL5.The remaining tissues were fixed, embedded, and sliced to identify Ki-67 expression and F4/80+CD206+ cell infiltration in tumor tissues.For analysis of the role of 12C8 in tumor treatment, an in vivo test was performed by administering 12C8 (3 mg kg −1 ) intravenously three times, with an equal amount of IgG used as a control.
Statistical Analysis: The data in this study were analyzed using Graph-Pad Prism.Unpaired Student's t test and unpaired t test with Welch's correction with a two-tailed distribution were used to determine the statistical significance.Dot plots were used to present the data from at least three independent experiments (mean±SEM).Spearman correlation analysis was performed to assess the association between ZBTB32 expression and CCL5 expression or macrophage infiltration, as well as the correlation between CD147-K148me2 levels and M2-like TAM infiltration.The overall survival of NSCLC patients was calculated using Kaplan-Meier analysis and the log-rank test.The associations between CD147-K148me2 or CD147 levels and different clinicopathological parameters in NSCLC patients were analyzed using the Chi-square test or Fisher's exact test.The body weight and tumor volume of the mice and the 12C8 binding ability for the peptide K148me2 were analyzed by two-way ANOVA.A P value less than 0.05 was considered to indicate statistical significance.

Figure 2 .
Figure 2. NSD2 acts on CD147 to generate CD147-K148me2.A) The levels of CD147-K148me2 and CD147 were determined by western blot in NSD2 knockdown A549 and H460 cells.B) Structural model of the CD147-NSD2 complex in a cartoon diagram, with CD147 in green and the NSD2 SET domain in blue.CD147-K148 and NSD2-SAM are shown as sticks, and the distance between them is indicated by yellow dashed line.C) The co-localization of CD147 (red) and NSD2 (green) in NSCLC tissue was detected by immunofluorescence staining, scale bar, 25 μm.D)The interaction between CD147 and NSD2 was confirmed in A549 and H460 cells by co-IP assays, and IgG was used as a negative control.E) The interaction between CD147 and NSD2 was analyzed in the rWT and rK148R cells (A549 and H460) by co-IP assays, and IgG was used as a negative control.F) The expression of CD147, NSD2, and CD147-K148me2 was determined in the NSD2-overexpressing cells (A549 and H460).G) The expression of CD147, NSD2, and CD147-K148me2 was determined in the NSD2-overexpressing rWT and rK148R cells (A549 and H460).H) An in vitro methyltransferase assay was performed with the K148 peptide and recombinant NSD2 protein co-incubated in methyltransferase buffer containing the methyl donor SAM.The di-methylation of the K148 peptide was detected by an anti-CD147-K148me2 antibody (12C8).I) An in vitro methyltransferase assay was performed with recombinant NSD2 protein and His-CD147-WT or His-CD147-K148R co-incubated in methyltransferase buffer containing the methyl donor SAM.The di-methylation of the CD147 protein was detected by an anti-CD147-K148me2 antibody (12C8).

Figure 3 .
Figure 3. CD147-K148me2 increases CCL5 secretion and M2-like TAM migration.A) DEGs (fold change (FC) >1.5 and P < 0.05) among the control (NC), CD147-WT (WT)-and CD147-K148R (Mut)-overexpressing A549 cells were identified by RNA-seq.B) The top 20 DEGs in the NC versus WT and WT versus Mut comparisons were displayed, and CCL5 was identified as the gene with the most significant difference between the WT and Mut groups.C) The expression levels of CD147, CD147-K148me2, and CCL5 were determined in the rWT and rK148R cells (A549 and H460) by western blot analyses.D) The mRNA level of CCL5 was determined in the rWT and rK148R cells (A549 and H460) by RT-PCR (*P < 0.05; **P < 0.01; n = 3).E) CCL5 levels in

Figure 4 .
Figure 4. CD147-K148me2 facilitates CCL5 secretion in NSCLC cells by activating CyPA-CD147-p38 signaling.A) Temperature factor (B-factor) distribution and K148 location of the CD147 molecule (PDB ID: 3B5H).B-factor values range from blue (low B-factor) to red (high B-factor).B) Structural superposition of CD147 (gray) and CD147-K148me2 (orange) based on their respective complex docking models.Arrows indicate local conformational changes induced by di-methylation, and yellow dashed lines indicate polar contacts formed between amino acid residues.C) Close-up view of the interaction interfaces of the docked complex model between CyPA (pink) and CD147 (green) without (upper) or with (lower) K148me2.K148 (red) and amino

Figure 5 .
Figure 5. CCL5 expression is closely related to the regulation of the transcription factor ZBTB32.A) The transcription factors associated with CCL5 expression were screened in the WT versus Mut and NC versus WT groups using RNA-seq analysis.Thirteen DEGs were shared between the two groups, and ZBTB32 was the only transcription factor.B) The correlation between ZBTB32 and CCL5 expression was analyzed in the NC and WT groups using RNA-seq data (R = 0.96, P = 0.0019).C) The correlation between ZBTB32 and CCL5 expression was analyzed in the WT and Mut groups using RNA-seq data (R = 0.91, P = 0.012).D) CCL5 expression was determined in the ZBTB32-overexpressing cells (A549 and H460) by western blot.E) CCL5 expression was determined in the ZBTB32 knockdown cells (A549 and H460) by western blot.F) The binding site of ZBTB32 to the CCL5 gene promoter was analyzed with a relative profile score threshold >90% using JASPAR.G) Dual-luciferase reporter assays were performed after co-transfecting of the pGL3-Basic-CCL5 promoter (−1000-−1), pRL-TK, and plasmid ZBTB32 or its control into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; n = 3).H) Dual-luciferase reporter assays were performed after co-transfecting of pGL3-Basic with different truncated sequences of the CCL5 gene promoter, pRL-TK, and the ZBTB32 plasmid into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; ns, not significant; n = 3).I) The predicted binding sequence of the CCL5 gene promoter (−387bp-TTTACAGTGT-−378 bp) was deleted and mutated to generate the new plasmids CCL5-Del (−400-−1) and CCL5-Mut (−400-−1), respectively.J) Dual-luciferase reporter assays were performed after co-transfecting of CCT-WT (CCL5-Del/CCL5-Mut) with pRL-TK and the plasmid ZBTB32 or its control into HEK293T cells.The relative luciferase signal was detected using a Dual-Luciferase® Reporter Assay System (***P < 0.001; ns, not significant; n = 3).

Figure 6 .
Figure 6.CD147-K148me2-mediated CCL5 secretion is involved in the regulation of ZBTB32.A) The expression levels of ZBTB32 and CCL5 were analyzed in the rWT and rK148R cells (A549 and H460) with or without recombinant CyPA stimulation by western blot.B) The expression of CCL5 was analyzed in the ZBTB32 knockdown cells (A549 and H460) with or without recombinant CyPA stimulation by western blot.C) The expression of ZBTB32 and CCL5 in the A549 and H460 cells treated with or without recombinant CyPA or SB203580 was analyzed by western blot.D) The expression levels of ZBTB32 and CCL5 were analyzed in the rWT and rK148R cells (A549 and H460) treated with SB203580 or U-46619 by western blot.E)The localization of the ZBTB32 protein (red) was identified in the rWT and rK148R cells (A549 and H460) using immunofluorescence staining, scale bar, 5 μm.F) Nuclear and cytoplasmic protein extraction assays were performed to determine the nuclear expression and cytoplasmic expression of ZBTB32 with or without recombinant CyPA stimulation in the rWT and rK148R cells (A549 and H460).Lamin B1 and GAPDH were selected as controls for nuclear protein and cytoplasmic protein, respectively.

Figure 7 .
Figure 7. CD147-K148me2 promotes NSCLC progression by inducing M2-like TAM infiltration.A) The expression of CD147 was determined in the LLC-WT and LLC-K148R cells by western blot.B) The expression levels of CD147, CD147-K148me2, and CCL5 were determined in the LLC-WT and LLC-K148R cells by western blot.C) Image of tumor masses from the LLC-WT (n = 6) and LLC-K148R (n = 6) groups.D) The body weights of the mice in the LLC-WT and LLC-K148R groups were measured every two days (ns, not significant; n = 6).E,F) The volume (E) and weight (F) of the tumor masses were measured (**P < 0.01; ***P < 0.001; n = 6).G,H) The percentage of Ki-67+ cells was determined in tumor tissues from the LLC-WT and LLC-K148Rgroups by IHC staining, scale bar, 100 μm (G).Three fields from each mouse were used for statistical analysis (n = 18 for each group; ***P < 0.001) (H).I,J) F4/80+CD206+ cell infiltration was determined in tumor tissues from the LLC-WT and LLC-K148R groups by multi-color immunofluorescence staining, scale bar, 50 μm (I).Five fields from each mouse were used for statistical analysis (n = 30 for each group; **P < 0.01) (J).K,L) The expression of ZBTB32 and CCL5 in tumor tissues was determined by western blot (K), and statistical analysis was conducted (n = 6 for each group; *P < 0.05; **P < 0.01) (L).

Figure 8 .
Figure 8.The anti-CD147-K148me2 antibody inhibits NSCLC immunosuppression and progression by interrupting the CyPA-CD147 interaction.A) The flow chart for tumor treatment with 12C8 antibody.B) Image of tumor masses from the IgG (n = 6) and 12C8 (n = 6) groups.C) The body weights of the mice in the IgG and 12C8 groups were measured every two days (ns, not significant; n = 6).D,E) The volume (D) and weight (E) of the tumor masses were measured (*P < 0.05; n = 6).F,G) The percentage of Ki-67+ cells was determined in tumor tissues from the IgG and 12C8 groups by IHC staining, scale bar, 100 μm (F).Three fields from each mouse were used for statistical analysis (n = 18 for each group; ***P < 0.001) (G).H,I) The infiltration of F4/80+CD206+ cells was determined in tumor tissues from the IgG and 12C8 groups by multi-color immunofluorescence staining, scale bar, 50 μm (H).Five fields from each mouse were used for statistical analysis (n = 30 for each group; *P < 0.05) (I).